2,4-Dienoyl-CoA reductase (EC 1.3.1.34) is an auxiliary enzyme of beta-oxidation, and it participates in the metabolism of unsaturated fatty enoyl-CoA esters having double bonds in both even- and odd-numbered positions. In this article we describe the molecular cloning of the human gene for the 120-kDa isoform of mitochondrial 2,4-dienoyl-CoA reductase (DECR). The gene is approximately 30 kb and comprises 10 exons varying in size from 79 to 203 bp and 9 introns whose sizes vary from 95 bp to about 10 kb. The 5' UTR and 3' UTR are included in exons 1 and 10, respectively. The promoter region contains putative binding sites for several transcription factors, e.g., Sp1, AP-2, AP-4, and C/EBP, but no TATA box was found. Primer extension analysis and 5' RACE-PCR revealed variability in the length of the 5'-UTR, the longest being 72 bp. Through the use of FISH analysis on metaphase chromosomes with a genomic fragment of 2,4-dienoyl-CoA reductase, the gene was assigned to the chromosomal band 8q21.3.